Question

I want introduction, material, methods, procedures, resulting, discussions and one line of conclusion :Experiment of
Isolation of bacteria ( with the gram straining)
Course : plant pathology

Answer 1

GRAM STAINING

Introduction : The Gram stain is the most important staining procedure in microbiology. It is used to differentiate between grampositive organisms and gram negative organisms. Hence, it is a differentialstain. Gram negative and grampositive organisms are distinguished from each other by differences in their cell walls.

Materials Required:

1. Clean glass slides
2. Inoculating loop
3. Bunsen burner
4. Bibulous paper
5. Microscope
6. Lens paper and lens cleaner
7. Immersion oil
8. Distilled water
9. 18 to 24 hour cultures of organisms

Reagents:

1. Primary Stain         -     Crystal Violet
2. Mordant                 -     Grams Iodine
3. Decolourizer          -     Ethyl Alcohol
4. Secondary Stain    - Safranin   

Gram Stain Procedure

1. Place slide with heat fixed smear on staining tray.
2. Gently flood smear with crystal violet and let stand for 1 minute.
3. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle.
4. Gently flood the smear with Gram’s iodine and let stand for 1 minute.
5. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle. The smear will appear as a purple circle on the slide.
6. Decolorize using 95% ethyl alcohol or acetone. Tilt the slide slightly and apply the alcohol drop by drop for 5 to 10 seconds until the alcohol runs almost clear. Be careful not to over-decolorize.
7. Immediately rinse with water.
8. Gently flood with safranin to counter-stain and let stand for 45 seconds.
9. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle.
10. Blot dry the slide with bibulous paper.
11. View the smear using a light-microscope under oil-immersion.

Result:

Typical Gram-negative bacteria:

1. Bordetella pertusis, the causative agent of whooping cough
2. Salmonella typhi, the causative agent of typhoid
3. Vibrio cholera, the causative agent of cholera
4. Escherichia coli, the normally benign, ubiquitous, gut-dwelling bacteria

DISCUSSION. The Gram stain is the most widely used staining procedure in bacteriology. It is called a differentialstain since it differentiates betweenGram-positive and Gram-negative bacteria.

CONCLUSION: The Gram stain method is a rapid and cost-effective test that is also highly reproducible and readily available in many laboratories. These features make the Gram stain method a more desirable screening procedurefor bacterial vaginosis in a clinic population.